|Line 68:||Line 68:|
Revision as of 12:28, 30 September 2011
Our lab's version of the Geitz lithium-acetate transformation method.
- 1% Potassium Acetate (autoclaved)
SPORULATION Mark Hickman uses this protocol and regularly has sporulation efficiencies of >50%. 1. Grow a 5 mL overnight culture; dilute 1:50 in the morning and grow for 4 hours at 30°C (to log phase). 2. Pellet cells, wash in 1 mL of 1% potassium acetate, and resuspend in 3-4 mL of 1% potassium acetate. 3. Incubate at room temperature on a roller wheel for 3 day (can incubate longer, if desired). NOTE: When sporulating, if diploid is homozygous for an auxotrophic mutation, add that nutrient to the potassium acetate (about ¼ the amount listed for addition to SD media… any more and it could be used as a nitrogen source).
1% Potassium Acetate
- Grow a 5 mL overnight culture; dilute 1:50 in the morning and grow for 4 hours at 30°C (to log
- Pellet cells, wash in 1 mL of 1% potassium acetate, and resuspend in 3-4 mL of 1%
- Incubate at room temperature on a roller wheel for 1 day and then transfer to 30°C for 2 more days (can incubate longer, if desired).
- Check culture for spore formation on day 2 and continue checking until you see tetrads.
- Inoculate 50 ml of YPD with 500 μL of the YPD overnight culture in a 250 ml flask. The 500 µl volume is approximate, and depends on the density of the strain you inoculate.
- Grow in shaking incubator for about 3-5 hours.
- Turn on 42°C water-bath (for heat-shock) if it is not already on.
- Harvest the cells by centrifuging in Eppendorf centrifuge model 5810R at 4000rpm (3130 xg) for 5 min. Resuspend pellet in 25 ml of sterile water by vortexing briefly. Pellet again and then resuspend in 1 ml of 100 mM LiAc.
- Transfer cell suspension to a 1.5 ml eppendorf tube, centrifuge at 3,000 xg for 2 min in an Eppendorf 5418 centrifuge and discard supernatant by removing it with a pipette.
- Add 400 µl 100 mM LiAc and resuspend cells by pipetting up and down. Aliquot 50 μL into 1.5 ml tubes (1 for each transformation). Pellet cells (3,000 xg for 2 min) and remove supernatant by aspiration.
- Add 300 μL T mix to each eppendorf tube of cells. Per one transformation reaction add IN ORDER:
- 240 μL 50% PEG 3350
- 35 μL 1.0 M lithium acetate
- 25 μL 2 mg/ml sssDNA
- 50 μL sterile H20 and 20 μL of DNA (Note: You are aiming for a final concentration between 0.1-10 μg for plasmid DNA. Adjust your DNA and water amounts to add 70 μL of volume total)
- Vortex to resuspend cells.
- Incubate for 30 minutes at 30°C.
- Incubate tubes in a water bath at 42°C for 20-25 (up to 40) min. The time may need to optimized for your strain and transformation conditions.
- Microfuge at 3,000 xg for 15s, and remove transformation mix with a micropipette. (NOTE: If you are transforming cells with a drug resistance marker such as KanMX, NatMX, HygMX or selecting for 5-FOA resistance, DO NOT plate your cells now, you need to do a recovery step. See below.)
- Add 200 µL of sterile water to each tube and resuspend cells by pipetting it up and down as gently as possible if high efficiency is important.
- Plate your cells using glass beads to spread the cells. Add 3-4 glass beads to each plate that you will be using, add about 200μL of cells + water, and spread by shaking the plate horizontally. To ensure single colonies:
- Plate 150 µl of sterile water and add 20 µl cell suspension in one selection plate #1.
- Plate the remaining 180 µl in selection plate #2
- Incubate at 30 °C. Colonies should appear after 2-4 days.
Recovery step for drug resistance markers and 5-FOA
- If you are plating your cells onto plates with G418, clonNat, hygromycin, or 5-FOA (basically if you are trying to select for anything BUT ability to grow without a particular amino acid) you need to give your cells some time to recover and express the resistance marker after you've transformed them. This is done after you have removed the transformation mix but before you plate the cells. You have two options for recovery:
- Gently resuspend cells in 1ml of YPD. Put this tube at 30°C for 1-4 hours (with a tube clamp to keep the eppendorf from popping open). After recovery, spin down the cells, resuspend in sterile water, and plate as above.
- Gently resuspend cells in 1ml of YPD. Plate 200μL cuture onto a YPD plate. In the morning, replica plate this lawn of cells onto a selection plate. You should see colonies in 2-4 days. We usually save the rest of the cells resuspended in YPD overnight at 30°C (with a tube clamp to keep the eppendorf from popping open) and plate about 200μL of this onto a selection plate in the morning just as a back-up.
- We have found that plating onto YPD and replica plating the next day gives the best results. For 5-FOA transformations it seems crucial to do it this way, as allowing the cells to grow in liquid YPD for any amount of time allows cells with mutations in URA3 to arise and these are able to grow on 5-FOA.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- *Megan N McClean 13:25, 30 September 2011 (EDT): According to the Botstein lab, when sporulating, if diploid is homozygous for an auxotrophic mutation, add that nutrient to the potassium acetate (about ¼ the amount listed for addition to SD media… any more and it could be used as a nitrogen source). You may need to play with the concentration of nutrient that you add. So far, the McClean lab has only sporulated prototrophic strains using this protocol, so the first person to try it/get it to work with an auxotrophic strain, please leave a note here!
- *Megan N McClean 13:25, 30 September 2011 (EDT): To sporulate yeast cells need to be starved for nitrogen in the presence of a nonfermentable carbon source.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
Adapted from the Botstein lab protocol available at: http://www.princeton.edu/genomics/botstein/protocols/Sporulation_and_Tetrad_Dissection.pdf
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.